Yeast culture



Patented Nov. 9, 1943 YEAST CULTURE Alfred S. Schultz and Lawrence Atkin, Bronx, and Charles N. Frey, Scarsdale, N. Y., assignors to Standard Brands Incorporated, New York, N. Y., a corporation of Delaware No Drawing. Application April 9, 1940, Serial No. 328,694

2 Claims.

The invention relates to a method for promoting growth of yeast. More particularly; it pertains" to a procedure which brings about an effective utilization of the nitrogen content of a nutriment, and includes correlated improvements and discoveries whereby propagation of yeast is enhanced.

An object of the invention is to provide a method whereby a growth promoting factor deflciency in a medium for propagation of yeast may be corrected.

A further object of the invention is to provide a procedure in accordance with which an effective utilization of the nitrogen content of carbamid by yeast-may be accomplished.

An additional object of the invention is the provision of a method which enables yeast fully to assimilate the nitrogen content of carbamid and in a manner that may be readily, economically incorporating said factor thereinto in an amount which obviates such deficiency.

Other objects of the invention will in part be obvious and will in part appear hereinafter.

The invention accordingly comprises the several steps and the relation of one or more of such steps with respect to each of the others thereof, which will be exemplified in the process hereinafter disclosed, and the scope of the invention will be indicated in the claims.

Our studies relative to bios requirements of yeast have facilitated consideration of the nitrogen metabolism thereof. We have found that synthetic or highly purified bios materials having an insignificant quantity of assimilable nitrogen are utilizable in a medium which otherwise is synthetic, and that the growth obtained thereby will be directly proportional to the amount of easily assimilable nitrogen present, for example, ammonium sulfate. However, the non-availability of nitrogen in other types of nitrogen containing materials is evidenced by the absence of a significant growth.

In these studies we have found that growth promoting factor HE is uniquely associated with nitrogen metabolism. A Saccharomyces cerevisiae yeast gave a poor yield or growth when carbamid was used as the nitrogen source as compared with ammonium sulfate. An investigation disclosed that the yield was increased and a normal crop obtained with carbamid nitrogen when the amount of I13 was increased; This evidences, hence, that a medium containing carbamid as a nitrogen source and deficient in IIB, whereby full utilization of the nitrogen is not obtained, may be brought to a condition to give a full yield corresponding to the nitrogen present when an amount of HE is added in order to make up the deficiency.

In the practice of the invention a medium, 1. e., a wort or spirit mash containing a yeast assimilable carbohydrate and carbamid as a nitrogen source, may be prepared and an amount of growth promoting factor IIB incorporated therewith. The medium may then be inoculated with yeast and the yeast permitted to grow or propagate therein under suitable temperature conditions as, for example; about 30 C. After a suitable period of time the yeast may be separated and the amount thereof determined, thus showing the influence of the factor IIB upon the yield or growth. A wort prepared in the usual manner, especially a synthetic wort, has been found to be deficient in factor 1113 and not to give a full yield with commensurate nitrogen utilization when carbamid is employed as the nitrogen nutriment. However, when IIB in the form of a concentrate, the pure substance being not known, is incorporated into such a wort in an amount sufficient to make up the deficiency, there is a complete a utilization of the nitrogen content of the carbamid with an accompanying normal yield or crop of the yeast.

charomyces cerevisiae, and more particularly numbers 765, 2335, 4125 and 4225 of the American type culture collection, Strain Delft I, Centraalbureau voor Schimmel cultures and Luft II, Rasse XII, and Spc. 152' of the Institut fiir Garungsgewerbe.

Asillustrative of a manner in which the invention may be practiced, the following procedure is presented. The effect of growth promoting factor IIB upon the nitrogen metabolism of yeast when employing carbamid as a nitrogen source may be shown'by preparing a medium containing dextrose, preferably chemically pure, a buffer-salt combination giving in solution a pH value of 5.0 to 5.5, inorganic salts as potassium, magnesium, calcium and sodium salts, inositol, and IIA or beta alanine. In addition thereto 1113 in solution is incorporated therewith. This medium may then be placed in suitable vessels, as a 200 ml. conical flask in an amount of 30 ml., and inoculated with 1 mg. of moist yeast. The medium containing yeast is then maintained at a temperature of about 30 C. with active shaking for a period which may be about 24 hours. At the end of this period from 1 to 2 m1. of the yeast suspension may be removed and have added thereto 2 ml. of a 10% solution of chloracetic acid. After standing for about 5 minutes the volume is made up to about ml. and centrifuged in Hopkins vaccine tubes. After about 5 minutes centrifuging at high speed, the volume of yeast sediment is read. The yield or crop values are calculated by taking the yeast volume equivalent to 10 ml. of yeast suspension and multiplying by 1000. By this determination 1 gram of moistyeast in the 30 ml. total volumeof growth medium gives a crop value of 222. The buffer solution, for example, may be prepared by dissolving potassium citrate and citric acid in water in suitable proportions to give a volume of 1 liter.

It was thus shown that when'IIs was added to a growth medium withoutany added nitrogen source there was substantially no influence upon the growth as shown by a crop value of 3. However, when the growth medium containted 1 gram carbamid and 1 ml. of 11s solution, the crop value was 150. Further, when the amount of Ha solution was doubled, i. e., when 2.0 ml. were added, the crop value was 200. This evidences a marked increase in crop value through the addition of a larger amount of th 11s solution, i. e., the 11s factor was that which limited the crop value when carbamid was included as the {nitrogen nutriment. Moreover, it is thus shown that whereas the carbamid nitrogen was not fully utilized in the first instance due to a deficiency of 11a, there was such a utilization when this deficiency was corrected through the incorporation of a larger quantity of the factor 11s. A like series of studies showed that with ammonium sulfate as the nitrogen source there is not a like action of the factor Ha.

The comparative effects of growth promoting factors HA or inositol, and Ha are evidenced also in the following tabulation. The yeast was grown in the manner heretofore set forth with a variation in the medium content only with respect to HA and Us.

Us solution Crop Nitrogen source [IA solution M illllile" M lllili The foregoing depicts that factor 11a is unique-.

ly associated with nitrogen metabolism of yeast when carbamid constitutes the nitrogen source. It is thus demonstrated also that utilization of nitrogen from carbamid requires more 11s but less of the factor IIA than does an analogous crop when using ammonia nitrogen, i. e., ammonium sulfate. As will be observed a low about 30C., and in theimannerhereinbefore 'described. Without any addition. of :iactor i-IIsithe.

yield of yeast as moist yeastcontainim.'27.%. solids and based upon the amount of :molaaes equivalent to the corn sugar :used :was 11%. When 50 cc. of about a il;Balling IIs'solution-was introduced the yield was 70%. "In the flrst'instance, due to factor IIa deficiency, the yeast'was not able to utilize the nitrogen present inxthe carbamid, although the amount. employed was suflicient in nitrogenv content to give a normal yield of the yeast, as is showncby the low yield obtained, whereas in: the second, when such denciency had been obviated,:the yield was-a normal amount of Ila more sharply reduces the carba- 7 one.

We accordingly have foundetbat'thesnitrogen metabolism of yeast when using carbamid asa nitrogen sourceis distinctly associated-with and influenced by growth promoting factor II: so-

that a medium giving-alow yieldof yeast'with carbamid as the-nitrogen source,.and beingdeflcient in growth promoting factor may be brought to a fullzutilisation ofwthernitrogen. con tent with attending increased; yield ofiyeast-by. incorporating. factor 113: inv an amount. snmcient to overcome thedeficiency. Thefa'ctor 11a may be produced byan extraction of cane distillery slop with butyl alcohol, methyl isobutyl ketone or ethyl ether at ap'H- which isacid ,tQCongo-red.

The preparation may also be brought. aboutb! concentrating molasses slop to 40' JB'alling and precipitating salts bythe addition of alcohol in an amount of about 8 0%. :The alcohol .is then evaporated; thev residue taken 311p with water, and an activated carbon introduced .thereinto at a pH acid to Congo red., Theactivatedcarbon adsorbs the factor and .this factorcmaylbe separated. therefrom by elutriation with, alcohol. Followlng'distillationof the alcohol, the concentrated IIa residue may be extracted .with ethy ether or methyl isobutyl. ketone and the He obtained therefromby washing withv water.

Since certain changes may bemadeincarryina out the above process without departing from the scope of the invention, it is intended that all.

matter contained in the above description shall be interpreted asillustrative andnot in a limiting sense.

Having described our invention, whatwe claim as new and desire to secure by Letters Patent is: 1. A method for promoting growth of yeast, which comprises incorporating growth promoting factor IIa into a wort containing a saccharomyces cerevisiae yeast and carbamid as a nitrogen source, in anamcunt suflicient to bringabout an efliclent utilization of the carbamid nitrogen: said amount of factor II: beingin exeessof that required to give an emcient utilization of ammonia nitrogen. 2. A method for promoting growth of yeast, which comprises propagating a 'Sacchmmgces cerevisiae yeast in a medium'ccntainlng a yeast assimllable carbohydrate, carbamld as the nitrogen source, and growth promoting factor Ha in an amount sumcient to bring about an eflicient utilization of the carbamid nitrogen; said amount of factor IIa being in excess of that required to give an efiicient utilization of ammonia'nitrogen.

ALFRED S. SCHULTZ. LAWRENCE AI'KIN. CHARLES N. FREI. 

